Proteins containing clusters of non-heme iron and inorganic sulfur are the most ubiquitous electron carriers in nature. They are intimately involved in such fundamental biological processes as respiration, photosynthesis, fermentation, and nitrogen fixation. It has recently been recognized, however, that iron-sulfur clusters fulfill functions other than simple electron transfer: they participate in binding small molecule substrates and catalyzing chemical reactions. Several metalloenzymes are now known that contain unusual iron-sulfur clusters as part of their catalytic sites. These include nitrogenase, hydrogenase, CO dehydrogenase, aconitase, and various hydrolases. The research proposed here is therefore designed to elucidate the mechanisms by which conventional iron-sulfur clusters in redox proteins have been "functionalized" such that they are able to take part in fundamental biological reactions in metalloenzymes. One hypothesis that has gained credence from the characterization of the active sites of metalloenzymes and the synthesis of analog compounds is that some or all of these "catalytic iron-sulfur clusters" are based on a cubane-type [Fe4S4] structural unit, that has been "functionalized" by non-cysteinyl ligation of a specific Fe site or by replacement of one Fe by another transition metal M. The overall objective of this proposal is to test this hypothesis by forming and investigating the spectroscopic and ligand binding properties of a [MFe3S4] cluster (where M = Fe, Ni, Mo, V, W) in a novel ferredoxin obtained from the extremely thermophilic bacterium, Pyrococcus furiosus, an organism that grows optimally at 100oC. P. furiosus ferredoxin (Mr = 7,500) contains a single [Fe4S4] cluster. The protein is remarkable both for its extreme thermal stability (it is stable for 95%C for at least 12 hours) and in being the only example of a 4Fe- ferredoxin that has non-cysteinyl ligation of one Fe atom, as evidenced by the novel spectroscopic properties of the [Fe4S4]2+1+ cluster and the ease of quantitative removal of this Fe atom to yield a conventional [Fe3S4]1+0 cluster. The properties of this ferredoxin make it the ideal system for investigating how iron-sulfur clusters may be functionalized. Using biochemical and recombinant DNA techniques in conjunction with an array of spectroscopies, e.g. electron paramagnetic resonance, magnetic circular dichroism, resonance Raman, Mossbauer, electron nuclear double resonance and X-ray absorption, the specific aims of this proposal are to investigate a) exogenous ligand binding to its [Fe4S4] cluster, b) the formation and characterization of mixed metal [MFe3S4] clusters, c) the structural consequences of mutations of specific amino acid residues, and d) the factors responsible for the "hyperthermostability" of the protein. In addition to providing insight into the structure, function and chemical properties of the Fe-S clusters in a broad range of metalloenzymes, the proposal also addresses the fundamental problems of metal cluster-protein interactions and protein-protein interactions in terms of stability and reactivity.